Raman spectroscopy is a powerful method for obtaining a chemical signature delineating the internal molecular structure of a sample. The applications and principles of Raman spectroscopy are well known and thus will not be described here in detail. Raman spectroscopy is an in-elastic light scattering technique that that uses the Raman effect. In a typical Raman spectroscopy system, an excitation laser illuminates a sample, for example a molecular or cellular sample, containing various molecules that provide the Raman scattering signal. The light that is scattered from the sample exhibits a frequency shift that reflects the energy of specific molecular vibrations within the sample. This provides a detailed chemical composition of the sample—a chemical fingerprint.
Because it may be applied to samples over a wide size range from single cells through to intact tissue, Raman spectroscopy has significant potential in biomedical science, e.g. in the early detection of disease. However, a major challenge of Raman spectroscopy is that the signal is very weak and may be masked by background fluorescence that is generated from components within the optical arrangement. as well as the sample. In particular, fluorescence can be generated from coatings on the surface of the optical components and/or within the optical components themselves. Considerable effort has been made to enhance the ratio of signal to background noise for Raman spectroscopy. Some benefits can be achieved by increasing the acquisition time typically to several minutes. However for live cells, long acquisition times can cause damage due to extended irradiation by the Raman excitation laser.
FIG. 1 shows a typical spectroscopy microscope setup 2 including a light source 4, an objective lens 6 for focusing light from the source onto a sample plane 8, a collimator 10 to collimate the light from the sample plane 8, and an additional lens 12 to focus the collimated light onto a detector 14 that measures the spectrum. The Raman signal will be generated at the sample plane 8 and efficiently collected onto the detector 14. Additionally fluorescence generated from optical system will be collected with varying efficiency. The path of fluorescent light generated from one specific point in the objective lens is shown. The amount of fluorescence collected and detected depends on the total fluorescence initially generated, the distance from the paraxial axis and the distance from the focal plane. The closer the fluorescence point is to the paraxial axis and/or to the focal plane the more fluorescence is detected. The amount of fluorescence detected is also dependant upon the intensity of the excitation, the beam profile and the collection efficiency. For example, when exciting with a Gaussian beam profile, the peak intensity is centrally situated causing the largest amount of fluorescence to be generated down the centre of the paraxial axis, allowing it to be efficiently collected by the detector. This is a problem.